Dataset Information

Chemical Proteomics Partners identification of Cycloamphilectene

ABSTRACT: The identification of the cycloamphilectene cellular interactome has been performed, through chemical proteomics, to unambiguously define its mechanism of action and thus to understand its pharmacological effect. To achieve this goal, a small bioactive molecule (cycloamphilectene) linked to a matrix through a spacer arm “fishes out” its specific interactors from a cell lysate or a tissue extract. Once eluted, cellular partners are separated by SDS-PAGE and identified by high resolution MS and bioinformatics analysis . MS and MS/MS data were acquired using a Q-TOF Premier mass spectrometer (Waters Corp., Micromass, Manchester, United Kingdom). The five most intense ions were automatically chosen by the MassLynx (4.1) software and fragmented. After mass spectrometric measurements, data were automatically processed by ProteinLynx Global Server software to generate peak lists for protein identifications. Database searches were carried out with MASCOT server. The SwissProt human database (release 2010_11 of 02 Nov 10, 522019 sequences, 184241123 residues) was searched, allowing 2 missed cleavages, carbamidomethyl (C) as fixed modification o and oxidation (M) and phosphorylation (ST) as variable modifications. The peptide tolerance was set to 80 ppm and the MS/MS tolerance to 0.8 Da. The Raw data reported herein contains 4 different chemical proteomics experiments carried out to identify cycloamphilectene cellular partner.

INSTRUMENT(S): Q-Tof Premier

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Luigi Margarucci

LAB HEAD: Prof. Agostino Casapullo

PROVIDER: PXD000106 | Pride | 2018-11-26

REPOSITORIES: Pride

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Publications

N-Formyl-7-amino-11-cycloamphilectene, a marine sponge metabolite, binds to tubulin and modulates microtubule depolymerization.

Margarucci Luigi L Monti Maria Chiara MC Esposito Roberta R Tosco Alessandra A Hamel Ernest E Riccio Raffaele R Casapullo Agostino A

Molecular bioSystems 20140204 4

The importance of protein-small molecule interaction in drug discovery, medicinal chemistry and biology has driven the development of new analytical methods to disclose the whole interactome of bioactive compounds. To accelerate targets discovery of N-formyl-7-amino-11-cycloamphilectene (CALe), a marine bioactive diterpene isolated from the Vanuatu sponge Axinella sp., a chemoproteomic-based approach has been successfully developed. CALe is a potent anti-inflammatory agent, modulating NO and pro ...[more]

PMID: 24493364

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Project description:Samples used in the study originated from three UK sites: the Walton Centre for Neurology and Neurosurgery in Liverpool, the Salford Royal Hospital in Salford and the Southern General Hospital in Glasgow. We recruited patients with pharmacoresistant mesial temporal lobe epilepsy for whom a therapeutic temporal lobectomy was being undertaken. After surgery, the hippocampus was divided into two portions: (1) one portion was preserved for RNA isolation, and (2) the other portion underwent histological analysis by an experienced neuropathologist. Frozen post-mortem histologically-normal hippocampal samples from donors with no known brain diseases were obtained from the MRC Edinburgh Brain Bank (Edinburgh, UK) and the Queen Square Brain Bank (London, UK). Brain samples were disrupted and homogenized in an appropriate volume of QIAzol lysis reagent (Qiagen, Crawley, United Kingdom) by using a TissueRuptor handheld rotor-stator homogenizer (Qiagen, Crawley, United Kingdom). Total RNA was extracted from the homogenates using the RNeasy Lipid Tissue Mini Kit (Qiagen, Crawley, United Kingdom), according to the manufacturer’s instructions. RNA quality was examined by capillary electrophoresis on an Agilent Bioanalyzer 2100 (Agilent, Palo Alto, CA) and Agilent 2100 Expert software was used to calculate the RNA Integrity number (RIN) of each sample. Purity of the RNA sample was assessed using a NanoDrop1000 Spectrophotometer. Capillary electrophoresis traces were also examined. Samples with RNA integrity number scores (RIN) below 6, obvious RNA degradation, significant 18S or 28S ribosomal RNA degradation, ratio of absorbance at 260nm and 280nm <1.95, or with noticeable DNA or background contaminants did not pass QC, and were withheld from microarray analysis. The microarrays were processed at the Centre for Genomics Research in the University of Liverpool (http://www.liv.ac.uk/genomic-research/). 50ng of total RNA was amplified and labelled using the Agilent Low Input Quick Amp One-Colour Labeling Kit and labelled RNA was hybridized to Agilent SurePrint G3 Custom Exon 8x60K Microarrays designed to contain probes for each exon of 936 selected genes, including all known SLC genes. Standard Agilent protocols were followed. One array failed on five of the QC criteria and, hence, was excluded. Intensity data were extracted from the remaining arrays using the Feature Extraction Software, in line with the manufacturer’s recommendations. The uploaded files contain data both for a custom exon array designed to contain probes for each exon of 936 selected genes and for a custom gene expression array designed to contain gene expression probes for genes across the whole genome.

Dataset Information

Chemical Proteomics Partners identification of Cycloamphilectene

Publications

N-Formyl-7-amino-11-cycloamphilectene, a marine sponge metabolite, binds to tubulin and modulates microtubule depolymerization.

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